CN107012155A - Participate in UGT genes and its coded product and the application of glycyrrhizic acid biosynthesis - Google Patents
Participate in UGT genes and its coded product and the application of glycyrrhizic acid biosynthesis Download PDFInfo
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Abstract
The invention discloses glycyrrhizic acid biosynthesis glycosyl transferase (UGT) gene and its coded product and application is participated in, belong to Gene Engineering of Medicinal Plants field.The gene obtains to be cloned first from radix glycyrrhizae, is the key gene of glycyrrhizic acid metabolic pathway of synthesizing, can directly be catalyzed generation glycyrrhizic acid.Nucleotide sequence provided by the present invention is as representated by SEQ ID No.1, and the protein sequence that it is encoded is as representated by SEQ ID No.2.The gene that the present invention is provided and glycyrrhizic acid biosynthesis are closely related, significant for glycyrrhizic acid content in regulation plant and artificial production glycyrrhizic acid.
Description
Technical field
The present invention relates to related glycosyl transferase (UGT) gene of glycyrrhizic acid biosynthesis, and utilize external enzymatic reaction
Method functional activity analysis is carried out to the gene coded protein, be related in glycyrrhizic acid biosynthesis pathway UGT genes and its
Coded product and application, belong to Gene Engineering of Medicinal Plants field.
Background technology
Glycyrrhizic acid is a kind of triterpene saponin natural products for deriving from Glycyrrhiza (Glycyrrhiz) plant, and it has wide
General bioactivity, including anti-inflammatory, antiallergy and antiviral etc..Current glycyrrhizic acid has been developed that into parenteral solution and clinically used
In the treatment of chronic hepatitis B and acute liver damage.It is reported that what is be made by main component of glycyrrhizic acid becomes a fine day sweet and refreshing injection year
Sales volume is up to 1,600,000,000 RMB.Simultaneously as the sugariness of glycyrrhizic acid is 150 times of sucrose, it also serves as sweetener and finds a good sale in the world
Various regions, its associated outlet product year sales volume is up to 4,000 ten thousand dollars, the huge market demand.
The biosynthesis of glycyrrhizic acid needs the relevant enzyme in its biosynthesis pathway to carry out a series of catalytic reactions in plant,
At present, the gene of most of enzymes in glycyrrhizic acid biosynthesis pathway is by successful clone, and the method that can utilize biotechnology
Artificial synthesized enoxolone, still, the UGT genes of the final step reaction of catalysis glycyrrhizic acid synthesis still fail by successful clone.
Therefore, clone glycyrrhizic acid UGT genes will be for adjusting and producing glycyrrhizic acid significant.
The content of the invention
(1) the problem to be solved in the present invention
UGT genes are important superfamily genes in plant, and member is numerous, vdiverse in function, clones and characterize its function past
It is past very difficult.Before the present invention comes forth, not yet there is any disclosure or reported and be related to glycyrrhizic acid UGT in present patent application
Nucleotides and its amino acid sequence.It is an object of the invention to provide glycyrrhizic acid UGT nucleotides and its amino acid sequence.
(2) technical scheme
To obtain glycyrrhizic acid UGT nucleotides and its amino acid sequence, we will clone obtained radix glycyrrhizae (Glycyrrhiza
Uralensis Fisch) in UGT nucleotide sequences be connected to expression vector and in host cell expression obtain albumen, then
By its function of catalysed in vitro Experimental Characterization, the present invention is finally realized.
The present invention is described in detail below.
[1] a kind of polynucleotides, are the polynucleotides shown in following (a), (b) or (c)：
(a) a kind of nucleotides, its nucleotide sequence is made up of 1-1485 nucleotides shown in SEQ ID No.1；
(b) a kind of polynucleotides, it is included has 35% or greater percentage homogeneity with nucleotide sequence (a) described
Nucleotide sequence, and the protein encoded has glycyrrhizic acid UGT activity；Or
(c) a kind of polynucleotides, its nucleotide hybridization under strict conditions with (a) reverse complemental, and the egg encoded
White matter has glycyrrhizic acid UGT activity.
[2] a kind of protein, is the protein shown in following (A), (B) or (C)：
(A) a kind of protein, its amino acid sequence is made up of the amino acid shown in SEQ ID No.2；
(B) a kind of protein, its amino acid sequence is included has 30% or higher percentage with the amino acid sequence described in (A)
Than the amino acid sequence of homogeneity, and with glycyrrhizic acid UGT activity；Or
(C) a kind of protein, its amino acid sequence, which is included in the amino acid sequence described in (A), has one or several amino
Missing, displacement, addition or the amino acid sequence of insertion of acid, and with glycyrrhizic acid UGT activity.
[3] a kind of recombinant vector, its as the polynucleotides described in [1] or coding [2] described protein polynucleotides with
Known carrier restructuring is formed.
[4] a kind of transformant, it is by will be according to the polynucleotides or coding described in the recombinant vector described in [3], [1]
[2] polynucleotides of protein described in are imported to be prepared from host.
[5] it is known cell system carrier or known cell free system carrier in the known carrier described in [3].
[6] the host described in [4] be belong to corynebacterium (Corynebacterium), general Pseudomonas (Pantoea),
Microorganism known to Enterobacter (Enterobacter) or Blastocystis (Saccharomyces).
[7] a kind of method for producing glycyrrhizic acid, methods described is by cultivating the conversion any one of [4] and [6]
Body directly produces glycyrrhizic acid.
[8] containing the polynucleotides or the polynucleotides of coding [2] described protein described in [1] in radix glycyrrhizae genetic breeding
Application.
(3) beneficial effect
Before the present invention comes forth, not yet there is any disclosure or reported and be related to glycyrrhizic acid UGT cores in present patent application
Thuja acid and its amino acid sequence, the invention provides glycyrrhizic acid UGT polynucleotide sequence, the albumen that they are encoded has radix glycyrrhizae
Sour UGT activity, and can be used for adjusting and producing glycyrrhizic acid.
It is of the present invention that " glycyrrhizic acid UGT " refers to thering is catalysis enoxolone (CAS：471-53-4) generation radix glycyrrhizae time
Sour list glucuronic acid (CAS：34096-83-8) or glycyrrhizic acid (CAS：1405-86-3), or catalysis enoxolone list grape
Uronic acid (CAS：34096-83-8) generation glycyrrhizic acid (CAS：UGT 1405-86-3).Described CAS refers to material numeral
Identification number (Chemical Abstracts Service), is the unique identification of the chemical substance in each natural world
Code.
Percentage identity of the present invention refers to by BLAST (Basic Local Alignment Search
Tool) algorithm is come the similitude between the nucleotides or protein that determine, and the percentage identity between nucleotides can pass through
BLASTN algorithms, which are calculated, to be determined, the percentage identity between protein can be calculated by BLASTP algorithms and determined, BLASTP and
BLASTN program can be found in website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Brief description of the drawings
Fig. 1 is glycyrrhizic acid UGT catalysis schematic diagrames；
Fig. 2 is that (M represents albumen marker, and 1 represents crude protein, and 2 represent pure for the electrophoretograms of SEQ ID No.1 encoding proteins
Change albumen, 3 represent the purifying protein after concentration)；
SEQ ID No.1 encoding proteins catalytic reaction products LC-MS chromatograms (figure when Fig. 3 is using enoxolone as substrate
Middle retention time is enoxolone for 5.84min peak, and retention time is enoxolone list grape alditol for 5.18min peak
Acid, retention time is glycyrrhizic acid for 4.46min peak)；
SEQ ID No.1 encoding proteins catalytic reaction products LC- when Fig. 4 is using enoxolone list glucuronic acid as substrate
(retention time is enoxolone list glucuronic acid for 5.18min peak to MS chromatograms in figure, and retention time is 4.46min's
Peak is glycyrrhizic acid).
Embodiment
The polynucleotides for the coding glycyrrhizic acid UGT that the present invention is obtained by clone are simultaneously connected to expression vector, then will
The recombinant expression carrier of polynucleotides with coding glycyrrhizic acid UGT is transferred in host respectively expresses.Extract obtained purifying egg
White be added in the external enzymatic reaction system containing different substrates is reacted, and catalysate is analyzed with LC-MS.LC-MS is tied
Fruit proves that the protease of the polynucleotide encoding has glycyrrhizic acid UGT activity.Particular content is as described below.
<Encode glycyrrhizic acid UGT polynucleotides>
The invention provides coding glycyrrhizic acid UGT polynucleotides.
In one embodiment, nucleotides of the invention is by the 1-1485 in SEQ ID No.1 nucleotide sequences
Position nucleotides composition, the amino acid sequence of its encoding proteins is as shown in SEQ ID No.2, and the protein has glycyrrhizic acid
UGT activity.
In another embodiment, polynucleotides of the invention are comprising the nucleotide sequence with SEQ ID No.1
There are the polynucleotides of the nucleotide sequence of 35% or greater percentage homogeneity, and the polynucleotides can encode and be respectively provided with glycyrrhizic acid
The protein of UGT activity.The percentage identity can be 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%,
92%th, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,
99.6%th, 99.7%, 99.8%, 99.9% or higher.
In another embodiment, polynucleotides of the invention be under strict conditions can be reverse with SEQ ID No.1
The polynucleotides of complementary nucleotide sequence hybridization, and the polynucleotides can encode the protein for being respectively provided with glycyrrhizic acid UGT activity.
" stringent condition " refers to wherein form condition of the so-called specific hybrid without forming non-specific hybridization.So
Condition be difficult to it is clearly quantitative.However, for example, the condition is that wherein substantially the same polynucleotides have height
The condition of homogeneity, for example, the polynucleotides phase mutual cross with percentage identity described above, and with relatively low same
The polynucleotides of one property do not hybridize mutually.Especially, the condition may include at about 45 DEG C in 6 × SCC (sodium chloride/citric acid
Sodium) in hybridize, then washed once in 0.2 × SCC and 0.1%SDS at 50 to 65 DEG C or twice or repeatedly.
The polynucleotides of the present invention can be DNA or RNA, and preferably DNA.The polynucleotides of the present invention can also be volume
The polynucleotides of the protein of code book invention hereafter.
<Glycyrrhizic acid UGT albumen>
Present invention also offers the protein with glycyrrhizic acid UGT activity.
In one embodiment, the amino acid sequence of protein of the invention is as shown in SEQ ID No.2, and it has sweet
Oxalic acid UGT activity.
In another embodiment, protein of the invention is that have comprising the amino acid sequence with SEQ ID No.2
30% or the protein of greater percentage homogeneity, and the protein is respectively provided with glycyrrhizic acid UGT activity.The percentage identity can
Be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%th, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more
It is high.
In still another embodiment, protein of the invention is included in SEQ ID No.2 amino acid sequence and had
There are the protein of one or several amino acid mutations, and the protein is respectively provided with glycyrrhizic acid UGT activity.Amino acid residue mutation
Example may include missing, displacement, addition and the insertion of one or several amino acid residues.Term " one or several " refers to egg
White three-dimensional structure and the activity of protein be not by the scope of substantial effect.
In the protein of the present invention, the site that imported into catalyst structure domain can will be mutated and in addition to catalyst structure domain
Site in, and keep original activity.The position that the amino acid residue to be mutated of purpose activity can be kept is this area skill
Known to art personnel.Especially, those skilled in the art can recognize the association between 26S Proteasome Structure and Function, because art technology
Personnel can：(1) amino acid sequence of active multiple protein with same type is compared (for example, by SEQ ID No.2
The comparison of the amino acid sequence of representative and other glycyrrhizic acids UGT amino acid sequence), (2) clear and definite conservative region relatively and non-
Conservative region relatively, (3) from relatively conservative region and non-conservative region relatively respectively predict can critical function
The region of effect and the region that can not be played an important role.Therefore, those skilled in the art result in glycyrrhizic acid UGT amino acid
The position of the amino acid residue to be mutated of sequence.
The displacement of amino acid residue can be conservative substitution or not influence the non-conservative displacement of protein active.
As used herein, term " conservative substitution " refers to residual using some amino acid of radical amino acid replacement with similar side chain
Base.The family of amino acid residue with similar side chain is as known in the art.The example of such family may include have
The amino acid (for example, lysine, arginine, histidine) of basic side chain, the amino acid with acid side-chain is (for example, asparagus fern ammonia
Acid, glutamic acid), the amino acid with uncharged polar side chain is (for example, asparagine, glutamine, serine, Soviet Union's ammonia
Acid, tyrosine, cysteine), amino acid with non-polar sidechain (for example, glycine, alanine, valine, leucine,
Isoleucine, proline, phenylalanine, methionine, tryptophan), in the amino acid with β positions with side chain (for example, Soviet Union
Propylhomoserin, valine, isoleucine), the amino acid with beta-branched side is (for example, tyrosine, phenylalanine, tryptophan, group ammonia
Acid), the amino acid (for example, serine, threonine, tyrosine) with hydroxyl (for example, alcohol of formula, phenolic) side chain, and have
The amino acid (for example, cysteine, methionine) of sulfur-containing side chain.Preferably, the conservative substitution of amino acid can be asparagus fern ammonia
Displacement between acid and glutamic acid, the displacement between arginine, lysine and histidine, putting between tryptophan and phenylalanine
Change, the displacement between phenylalanine and valine, the displacement between leucine, isoleucine and valine, and glycine and third
Displacement between propylhomoserin.
<Recombinant vector>
The invention provides recombinant vector.The recombinant vector of the present invention is in the polynucleotides or the coding present invention of the present invention
The polynucleotides of protein are formed with known carrier restructuring.Described known carrier may include cell system carrier, and (it is in host
Marking protein in cell) and cell free system carrier (it utilizes protein translation system).Known carrier can also be plasmid
Or integration vector.Known carrier can also be DNA vector or RNA carriers.
The example of cell system carrier includes being based on ColE plasmids, pBR322 derivatives is represented as, with p15A starting points
Based on pACYC plasmids, plasmid based on pSC and from Bac the F factors small-sized F plasmids and Escherichia coli
Similar expression vector in (Escherichia coli).In addition, it may further comprise with trp promoter such as trc and tac
Expression vector, lac promoters, T7 promoters, T5 promoters, T3 promoters, SP6 promoters, arabinose-inducible start
Son, cold shock promoters, tetracycline inducible promoter and analog.
The example of cell free system carrier may include to have the T7 promoters of example or T3 in cell system carrier to start
The expression vector of son and the carrier for the synthetic protein in wheat cell free system, such as the plasmid based on pEU, it has
SP6 promoters or T7 promoters.
In using the synthesis of the protein of cell free system carrier, first with re-recording system by the cDNA of target protein
Transcribe and synthesize mRNA.The re-recording system may include routinely and publicly those known re-recording systems, wherein passing through RNA
Polymerase transcription cDNA.The example of RNA polymerase may include t7 rna polymerase.
Therefore, mRNA is translated with synthetic protein using cell-free protein synthesis system, the system is translation system.
In the system, closed comprising the ribosomes needed for translation, translation initiation factor, translation elongation factor, dissociation factor and aminoacyl tRNA
Into enzyme.The example of such protein translation system may include that E. coli extract, rabbit granulophilocyte and wheat embryo carry
Take thing.
Also, the cell-free protein synthesis system of reconstruct is may also include, the factor needed for it is translated more than is constituted, its
Independently purify.
The protein synthesis carried out using cell system carrier will below<Transformant>Described in.
The protein synthesized using cell system carrier or cell free system carrier can be purified.The example of purification process can be wrapped
Include the method using salting-out method and a variety of chromatography methods.When design expression vector is to express sequence label such as target protein
C-terminal or N-terminal it is histidine-tagged, can apply using such as nickel or cobalt the matter utilization to the label with affinity
The purification process of affinity chromatography method.In addition, passing through appropriate purification process and ion-exchange chromatography, gel permeation chromatography or class
As method it is combined improve the present invention protein purity.
<Transformant>
The invention provides the transformant of the expression vector comprising the present invention.The transformant of the present invention is by by the present invention
Expression vector imported into a kind of transformant obtained in host.Host for the present invention is preferably bacterium or fungi.
Bacterium can be gram-positive bacterium or gramnegative bacterium.
Gram-positive bacteria may include to belong to the bacterium with subordinate：Bacillus (Bacillus), listeria
(Listeria), staphylococcus (Staphylococcus), streptococcus (Streptococcus), enterococcus spp
(Enterococcus), fusobacterium (Clostridium), corynebacterium (Corynebacterium) and streptococcus
(Streptomyces).Preferably belong to the bacterium of Bacillus and corynebacterium.
The bacterium of Bacillus may include bacillus subtilis (Bacillus subtilis), bacillus anthracis (Bacillus
) and bacillus cereus (Bacillus cereus) anthracis.Bacillus subtilis is preferred.
The bacterium of corynebacteria may include corynebacterium glutamicum (Corynebacterium glutamicum), effective rod
Shape bacillus (Corynebacterium efficiens) and corynebacterium callunae (Corynebacterium callunae).
Corynebacterium glutamicum is preferred.
The example of gramnegative bacterium may include to belong to the bacterium with subordinate：It is Colibacter (Escherichia), general
Pseudomonas (Pantoea), Salmonella (Salmonella), vibrio (Vivrio), Serratia (Serratia) and intestines
Bacillus (Enterobacter).It is preferred to belong to the bacterium of Colibacter and Enterobacter.
It is preferred as the Escherichia coli of Colibacter are belonged to.
Belonging to the example of the bacterium of general Pseudomonas may include the general bacterium of pineapple (Pantoea ananatis), the general bacterium of Si Shi
(Pantoea stewartii), pantoea agglomerans (Pantoea agglomerans) and citrea (Pantoea citrea).
The general bacterium of pineapple and citrea are preferred.The bacterial strain of example can be used as belonging to general bacterium in European Patent Application Publication 0952221
The bacterium of category.Belonging to the example of the representative bacterial strain of the bacterium of general Pseudomonas may include to be disclosed in European Patent Application Publication
The general bacterium AJ13355 bacterial strains (FERMBP-6614) of pineapple and the general bacterium AJ13356 bacterial strains (FERMBP- of pineapple in 0952221
6615)。
Belong to the example of the bacterium of Enterobacter may include enterobacter agglomerans (Enterobacter agglomerans) and
Clostridium perfringen (Enterobacter aerogenes).Clostridium perfringen is preferred.European Patent Application Publication 0952221
The bacterium bacterial strain of middle example can be used as belonging to the bacterium of Enterobacter.Belong to the example of the representative bacterial strain of the bacterium of Enterobacter
It may include enterobacter agglomerans ATCC12287 bacterial strains, clostridium perfringen TACC13048 bacterial strains, clostridium perfringen NBRC12010 bacterial strains
(Biotechnol.Bioeng., 2007, Mar.27；98(2)：340-348) with clostridium perfringen AJ110637 (FERM BP-
10955)。
The example of fungi may include to belong to the microorganism with subordinate：Yeast (Saccharomyces), fission yeast
(Schizosaccharomyces), Yarrowia sp (Yarrowia), trichoderma (Trichoderma), aspergillus
(Aspergillus), reaping hook mould (Fusarium) and Mucor (Mucor).Belong to saccharomyces, Schizosaccharomyces, Yarrow saccharomyces
Or the microorganism of trichoderma is preferred.
Belonging to the example of the microorganism of saccharomyces may include this primary yeast (Saccharomyces of karr
Carlsbergensis), saccharomyces cerevisiae (Saccharomyces cerevisiae), Disseminating yeast (Saccharomyces
Diastaticus), Doug Laplace yeast (Saccharomyces douglasii), Saccharomyces kluyveri (Saccharomyces
Kluyveri), promise ground yeast (Saccharomyces norbensis) and ellipsoideus yeast (Saccharomyces
oviformis).Saccharomyces cerevisiae is preferred.
Schizosaccharomyces pombe (Schizosaccharomyces pombe) is excellent as the microorganisms of Schizosaccharomyces is belonged to
Choosing.
Solution fat Yarrowia sp (Yarrowia lypolytica) as belong to Yarrowia sp belong to microorganism be
It is preferred that.
Belonging to the example of the microorganism of trichoderma may include Trichoderma harzianum (Trichoderma harzianum), Kang Shi wood
Mould (Trichoderma koningii), long shoot trichoderma (Trichoderma longibrachiatum), trichoderma reesei
(Trichoderma reesei) and Trichoderma viride (Trichoderma viride).Trichoderma reesei is preferred.
In addition, the host for the present invention is not limited to specifically, host can also be that microorganism, insect cell, animal are thin
Born of the same parents, plant cell, insect tissue, animal tissue, plant tissue, insect organ, animal organ, plant organ, insect are all, dynamic
Thing entirety, plant entirety or the like.
Embodiment
The present invention will be specifically described by representative of the SEQ ID No.1 in nucleotides sequence list below, but the present invention is not limited
In following examples.
Embodiment 1：UGT clone in Glycyrrhiza Uralensis
We extract radix glycyrrhizae (Glycyrrhiza uralensis by RNA extracts kits (Japanese TAKARA companies)
Fisch) total serum IgE in root, cDNA is obtained with Reverse Transcriptase kit (Japanese TAKARA companies), the 5 ' ends according to UGT gene orders
With 3 ' primers of the end design with restriction enzyme site, enter performing PCR with the HS taq enzymes (Japanese TAKARA companies) of high-fidelity and expand,
PCR primer is sequenced and spliced, and can obtain the total length nucleosides of the related UGT genes of glycyrrhizic acid biosynthesis of the present invention
Acid sequence, it is as representated by SEQ ID No.1, and the length of its total length opening code-reading frame (ORF) is 1485bp, encodes 494 ammonia
Base acid, the SEQ ID No.2 that detailed amino acid sequence is shown in sequence table.
Embodiment 2：The structure of recombinant expression carrier
We are by the glycyrrhizic acid UGT obtained in embodiment 1 nucleotide fragments (SEQ ID No.1) and expression vector pET-
32a (+) with restriction enzyme (Japanese TAKARA companies) while carry out double digestion, and the product after digestion is returned with Ago-Gel
The method for receiving kit (Japanese TAKARA companies) is purified and reclaimed, the gold of the product DNA ligase of recovery at 16 DEG C
Connect and stay overnight in category bath, obtain recombinant plasmid.Recombinant plasmid convert again bacillus coli DH 5 alpha competent cell and coated plate in containing
Screened in the solid medium of ampicillin, positive colony send sequence verification again, and correctly clone passes through matter to sequence verification
The method of grain purification kit (Japanese TAKARA companies) is recyclable to obtain the restructuring table with SEQ ID No.1 nucleotide sequences
Up to carrier.
Embodiment 3：The structure of Host Strains
The recombinant expression carrier obtained in embodiment 2 is converted specification (Japanese TAKARA by us according to competent cell
Company) method conversion e. coli bl21 (DE3) competent cell and coated plate in the solid medium containing ampicillin
Middle screening, positive colony send sequence verification again, and sequence verification is correctly cloned as Host Strains, and it contains with SEQ ID
The recombinant expression carrier of No.1 nucleotide sequences.
Embodiment 4：Induced expression and protein purification
The bacterium solution 2mL of the Host Strains obtained in embodiment 3 is separately added into 100mL LB fluid nutrient mediums by we,
Concussion and cultivate is to exponential phase under the conditions of 37 DEG C, now OD600Value about 0.6 to 1.0.Wherein not contain glycyrrhizic acid UGT's
The Host Strains of empty carrier pET-32a (+) conversion e. coli bl21s (DE3) are used as negative control.Above-mentioned 4 Host Strains add respectively
Enter 0.4mM isopropylthiogalactosides (IPTG), 18 DEG C, induce more than 12h.5000rpm, 4 DEG C of centrifugation 5min collect thalline,
With the distillation water washing 2 times of precooling, thalline is collected.Ultrasonication bacterium in 2ml distilled water suspension thalline again, ice bath is added,
Condition is：Ultrasonic 10s, is spaced 10s, totally 12 times.Whole operation is carried out in ice bath, and shaking bacterium solution frequently prevents it from overheating,
Ice bath is taken out every 3 times once.4 DEG C, 13000rpm centrifugation 2min collect supernatant.Supernatant passes through protein purification kit
The method of (Japanese TAKARA companies) purifies and obtains purifying protein (Fig. 2) with glycyrrhizic acid GUT and without glycyrrhizic acid GUT
Purifying protein.
Embodiment 5：The catalytic analysis of albumen
Obtained in embodiment 42 kinds of purifying proteins are added separately to enoxolone (CAS by we：471-53-4) and
Enoxolone list glucuronic acid (CAS：34096-83-8) to be reacted in the external enzymatic reaction system of substrate, catalysis is anti-
Answer the Tric-HCl (pH 8.0) of buffer system including 50mM, 1mM uridine -5- diphosphate glucoses aldehydic acid (UDP-GlcA),
1mM dithiothreitol (DTT) (DTT) and 200 μM of substrate enoxolones, catalytic reaction temperature be 30 DEG C, catalysis time on 30min,
Contain the catalystic converter system with glycyrrhizic acid GUT purifying proteins as catalysis group, containing without glycyrrhizic acid GUT purifying proteins
Catalystic converter system as a control group, catalysate is analyzed with LC-MS.LC-MS instruments are the efficient liquid of the pump of Accela 600
Phase chromatogram-LTQ-Orbitrap XL GC-MSs (Thermo Scientific companies of the U.S.), equipped with electric spray ion source
(ESI).Chromatographic column is Agilent Zorbax SB C18 posts (250mm × 4.6mm × 5 μm).Mobile phase A is 0.1% formic acid
Water, B is pure methanol.Linear gradient elution program is：0~3.5min, 79%B；3.5~8.2min, 79%~98%B；8.2~
11min, 98%~79%B；11~20min, 79%B.Flow velocity 1.0mL/min, 25 DEG C of column temperature, the μ L of sample size 10.Mass spectrum ESI is examined
Survey as anionic textiles, full ion scan pattern.As a result find, catalysis group generates new peak compared with control group, passes through
Retention time, accurate molecular quasi-molecular ions and the major cleavage mode of contrast standard product and new peak, it is determined that with enoxolone (CAS：
471-53-4) enoxolone list glucuronic acid (CAS is generated for the catalysis group (Fig. 3) of substrate：34096-83-8) and radix glycyrrhizae
Acid (CAS：1405-86-3)；With enoxolone list glucuronic acid (CAS：34096-83-8) produced for the catalysis group (Fig. 4) of substrate
Glycyrrhizic acid (CAS is given birth to：1405-86-3).Therefore, it is experimentally confirmed, nucleotides (sequence is as shown in SEQ ID No.1) coding
Albumen (sequence such as SEQ ID No.2) there is catalysis enoxolone (CAS：471-53-4) generate enoxolone list grape alditol
Acid (CAS：34096-83-8) or glycyrrhizic acid (CAS：1405-86-3), or catalysis enoxolone list glucuronic acid (CAS：
34096-83-8) generation glycyrrhizic acid (CAS：Activity 1405-86-3).
Embodiment 6：Mutation analysis
We are by the method for point mutation kit (Japanese TAKARA companies) by the partial amino-acid in SEQ ID No.2
Sequence carries out knockout experiment, and knock out site includes respectively：" E " (the now mutant nucleosides in the site of SEQ ID No.2 sequences 4
The percentage identity of acid sequence and SEQ ID No.1 nucleotide sequences is 99%, the mutein sequence and SEQ ID
The percentage identity of No.2 protein sequences for 99%), " EKQ " (the now mutation in the site of SEQ ID No.2 sequences 4 to 6
The percentage identity of body nucleotide sequence and SEQ ID No.1 nucleotide sequences is 99%, the mutein sequence with
99%) percentage identity of SEQ ID No.2 protein sequences is.Experiment proof is carried out by the method for embodiment 2 to 5：
The albumen that partial amino-acid series are knocked out in the present embodiment still has glycyrrhizic acid UGT activity.
We are also by the method for point mutation kit (Japanese TAKARA companies) by the part amino in SEQ ID No.2
Acid sequence enters line replacement experiment, and replacement site includes respectively：" D " in the site of SEQ ID No.2 sequences 120 is replaced as " A " (now
The percentage identity of the mutant nucleotide sequence and SEQ ID No.1 nucleotide sequences is 99%, the mutein
The percentage identity of sequence and SEQ ID No.2 protein sequences for 99%), the site of SEQ ID No.2 sequences 9 to 11
" KLK " is replaced as " EAD " (now percentage identity of the mutant nucleotide sequence and SEQ ID No.1 nucleotide sequences
For 99%, 99%) percentage identity of the mutein sequence and SEQ ID No.2 protein sequences is.Pass through reality
The method for applying example 2 to 5 carries out experiment proof：The albumen of part amino acid sequence substitutions still has glycyrrhizic acid UGT in the present embodiment
Activity.
We are also added the nucleotide sequence with maltose-binding protein MBP labels by the method for Gene Fusion respectively
5 ' ends of SEQ ID No.1 nucleotide sequences are added in, new nucleotide sequence is obtained, RHGS1 (now mutant cores are named as
74%) percentage identity of nucleotide sequence and SEQ ID No.1 nucleotide sequences is.Will by the method for embodiment 2 to 4
RHGS1 and carrier pMal-c4X, which are attached and expressed, obtains new fusion protein, is named as RHDB1 (now mutant eggs
74%) percentage identity of white matter sequence and SEQ ID No.2 protein sequences is.Divided by the method for embodiment 5
Analysis shows：The albumen RHDB1 that partial amino-acid series are added in the present embodiment still has glycyrrhizic acid UGT activity.
Nucleotide sequence with TrxTag label proteins is also inserted in SEQ by us by the method for Gene Fusion
The new nucleotide sequence obtained between 12 to 13 sites of ID No.1 nucleotide sequences, is named as RHGS2 (now mutant
82%) percentage identity of nucleotide sequence and SEQ ID No.1 nucleotide sequences is.Will by the method for embodiment 2 to 4
RHGS2 and carrier pMal-c4X, which are attached and expressed, obtains new fusion protein, is named as RHDB2 (now mutant eggs
White matter sequence and the percentage identity of SEQ ID No.2 protein sequences are divided for 82%) by the method for embodiment 5
Analysis shows：The albumen RHDB2 that partial amino-acid series are inserted in the present embodiment still has glycyrrhizic acid UGT activity.
Meanwhile, our also engineer's SEQ ID No.3 nucleotide sequences, itself and SEQ ID No.1 nucleotide sequences
Percentage identity for 35%, SEQ ID No.3 encoding proteins and SEQ ID No.2 amino acid sequences percentage identity
For 30%.Experiment proof is carried out by the method for embodiment 2 to 5：The albumen that SEQ ID No.3 are encoded in the present embodiment still has
Glycyrrhizic acid UGT activity.
Embodiment 7：Sequence hybridization is analyzed
We by SEQ ID No.3 and SEQ ID No.1 reverse complemental nucleotides at about 45 DEG C in 6 × SCC (chlorinations
Sodium/sodium citrate) in hybridized, then washed once in 0.2 × SCC and 0.1%SDS at 50 to 65 DEG C or twice or
Repeatedly.As a result show：Under this condition, two nucleotides successful cross.Meanwhile, SEQ ID No.3 have been proved in embodiment 6
The albumen of coding has glycyrrhizic acid UGT activity, so, the result is proved：If can be with SEQ ID No.1 reverse complemental nucleosides
Acid sequence hybridizes, and nucleic acid molecule of the coding with protein active glycyrrhizic acid UGT is also in the scope of the present invention.
Embodiment 8：Produce the structure of the Yeast engineering bacteria of glycyrrhizic acid
We by the method for embodiment 1 clone known bAS genes (Genbank number of registrations are GU072921.1),
(Genbank number of registrations are for CYP72A154 genes (Genbank number of registrations are AB558153.1), CYP88D6 genes
AB433179.1), (Genbank number of registrations are for AtCPR1 (Genbank number of registrations are NM_001203894.1) and UGDH genes
AJ007702.1), while we have also cloned known promoter (P in saccharomycetePGK1、PTEF1、PFBA1、PTDH3), terminator
(TADH1、TCYC1、TTPI1、TTDH2) and tHMGM1 genes.Then, by the method for Gene Fusion, we obtain fusion fragment
RH1(PPGK1-tHMG1-TADH1)、RH2(PTDH3-SEQ ID No.1--TTPI1-PTEF1-UGDH-TCYC1) and RH3 (PPGK1-
CYP72A154-TADH1-PFBA1-CYP88D6-TTDH2-PTDH3-AtCPR1-TTPI1-PTEF1-bAS-TCYC1).Then, we use
Three fusion fragments of acquisition are directed respectively into saccharomyces cerevisiae (Saccharomyces by the method for homologous recombination
Cerevisiae δ DNA sites, rDNA sites and His3 sites) in BY4742 strain genes group builds engineering bacteria.The work of structure
Journey bacterium is 7 days in yeast extract peptone glucose (YPD) agar medium, and condition of culture is 30 DEG C and 250rpm.Cultivate obtained production
Thing is detected with the method for embodiment 5, as a result proves to obtain 9.8mg/L glycyrrhizic acid.
Embodiment 9：Application of the SEQ ID No.1 genes in radix glycyrrhizae hairy root genetic breeding
The present embodiment describes conventional radix glycyrrhizae genetic improvement mode by taking the genetic improvement of radix glycyrrhizae hairy root as an example.According to SEQ
5 ' ends of ID No.1 gene orders and 3 ' primers of the end design with restriction enzyme site, respectively by SEQ ID No.1 genes and carrier
PBI121 carries out double digestion and recovery product, is stayed overnight with T4DNA ligases in 4 DEG C of connections, takes 1 μ L connection products to convert large intestine bar
Bacterium DH5 α, bacterium solution enters performing PCR inspection, and correct monoclonal bacterium solution is sequenced.After analyzing sequencing sequence, extract just
The plasmid really recombinated and transformed competence colibacillus Agrobacterium.Agrobacterium after conversion is screened in YEB solid mediums and tested with PCR
Card.PCR examines correct Agrobacterium to be used to infect radix glycyrrhizae (Glycyrrhiza uralensis Fisch) hairy root, after infecting
Radix glycyrrhizae hairy root be placed in new MS culture mediums 25 DEG C of cultures, be transferred in 1/2MS fluid nutrient mediums and cultivate afterwards, PCR identifications
The hairy root successfully infected.As a result show：Compared with control group, the radix glycyrrhizae hair of SEQ ID No.1 gene overexpressions is successfully transferred to
The content of glycyrrhizic acid improves 2.1 times in shape root.
Claims (8)
1. a kind of polynucleotides, are the polynucleotides shown in following (a), (b) or (c)：
(a) a kind of nucleotides, its nucleotide sequence is made up of 1-1485 nucleotides shown in SEQ ID No.1；
(b) a kind of polynucleotides, it includes the nucleosides for having 35% or greater percentage homogeneity with nucleotide sequence (a) described
Acid sequence, and the protein encoded has glycyrrhizic acid UGT activity；Or
(c) a kind of polynucleotides, its nucleotide hybridization under strict conditions with (a) reverse complemental, and the protein encoded
With glycyrrhizic acid UGT activity.
2. a kind of protein, is the protein shown in following (A), (B) or (C)：
(A) a kind of protein, its amino acid sequence is made up of the amino acid shown in SEQ ID No.2；
(B) a kind of protein, its amino acid sequence includes same with 30% or greater percentage with the amino acid sequence described in (A)
The amino acid sequence of one property, and with glycyrrhizic acid UGT activity；Or
(C) a kind of protein, its amino acid sequence, which is included in the amino acid sequence described in (A), has one or several amino acid
Missing, displacement, addition or the amino acid sequence of insertion, and with glycyrrhizic acid UGT activity.
3. a kind of recombinant vector, the multinuclear of its protein as described in the polynucleotides described in claim 1 or coding claim 2
Thuja acid is formed with known carrier restructuring.
4. a kind of transformant, it is by by the recombinant vector described in claim 3, the polynucleotides described in claim 1 or volume
The polynucleotides of protein are imported described in code claim 2 is prepared from host.
5. the known carrier described in claim 3 is known cell system carrier or known cell free system carrier.
6. the host described in claim 4 is to belong to corynebacterium (Corynebacterium), general Pseudomonas (Pantoea), intestines
The known microorganisms of Bacillus (Enterobacter) or Blastocystis (Saccharomyces).
7. a kind of method for producing glycyrrhizic acid, methods described is by cultivating according to claim 4 and appointing according in claim 6
Transformant described in one directly produces glycyrrhizic acid.
8. the polynucleotides of protein are educated in radix glycyrrhizae heredity described in the polynucleotides or coding claim 2 described in claim 1
Application in kind.
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| CN102796762A (en) * | 2012-09-11 | 2012-11-28 | 山东大学 | Application of arabidopsis glycosyl transferase gene UGT 76C2 in improving plant drought resistance |
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| CN102796762A (en) * | 2012-09-11 | 2012-11-28 | 山东大学 | Application of arabidopsis glycosyl transferase gene UGT 76C2 in improving plant drought resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371080A (en) * | 2018-10-15 | 2019-02-22 | 北京理工大学 | A kind of method of enzyme process preparation monosaccharide groups enoxolone Galactoside derivative |
| CN109371080B (en) * | 2018-10-15 | 2022-02-11 | 北京理工大学 | Method for preparing glycosyl glycyrrhetinic acid galactoside derivative by enzyme method |
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